The analysis of both the molecular composition and molecular activity within a particular substance is important in many different applications, including the detection of biomarkers for disease diagnosis and DNA sequencing, drug discovery and screening, and immunosignaturing, which involves detecting the changes of human antibodies.
One conventional system for detecting particular molecules within a substance involves introducing a fluorescent dye molecule into a particular sample. The dye marker is configured to bind to the molecules of interest, if present, within the sample. After introduction of the dye molecule, the sample is irradiated using ultraviolet (UV) radiation, which causes dye molecules that have bonded to the target molecule to fluoresce. If the sample fluoresces, that fluorescence indicates that the molecule of interest is present within the sample. Generally, sensitive optical instruments are used to monitor the sample for fluorescence and, thereby, identify the presence of the molecule of interest.
These fluorescence-based detection systems suffer from a number of drawbacks. First, the systems require UV light sources and highly sensitive optical detectors to detect the weak fluorescent light emission from a sample. This equipment can be very expensive and bulky. Additionally, when attempting to detect small molecules, the fluorescent dye molecule often alters the native properties of molecules of interest, rendering test results inaccurate and of little value. Finally, fluorescence detection is only useful as an end-point test and cannot be used to study the kinetics of molecular interactions and binding processes.
In response to the difficulties associated with fluorescent label analysis and other label analysis methods, label-free, optical detection technologies have been developed. One such method involves the analysis of data captured from the excitation of surface plasmons in a sample material. The instrumentation required to perform such surface plasmon resonance (SPR), however, is bulky and expensive due to the need of complex optics. This system also requires the fabrication of SPR chips (gold-coated glass chips), which is expensive and difficult.
Another label-free detection method relies upon detecting light interference created by a structured surface (e.g., multiple layer coating of chips) over which a sample is deposited. Although this approach may be somewhat simpler than SPR, it is also less sensitive than SPR. In some cases, to improve sensitivity, deep microstructures are etched onto a silicon chip to increase the surface area of the chip. This approach, therefore, introduces additional fabrication steps into the analysis process. Additionally, these microstructures can affect the molecule binding kinetics because, prior to binding, the molecules must diffuse into the constricted spaces defined by the microstructures.
The non-label, optical approaches called for in both SPR and interference-based analysis are all based upon measuring the optical mass of particular molecules. Because the optical mass of a molecule is proportional to the size of the molecule, these analysis techniques are not effective for detecting small molecules.
Another alternative for the molecular analysis of a sample involves the use of Microfabricated Electromechanical System devices (MEMS), such as cantilevers, tuning forks and quartz-crystal resonators, which can each be used for label-free detection of mass and spring constant changes associated with molecular binding events. In many cases, though, viscous damping of these mechanical devices, resulting from their placement within a sample aqueous solution, severely limits the ability of such systems to make accurate measurements. Additionally, the fabrication of MEMS devices requires microfabrication and cleanroom facilities, which are labor-intensive and expensive. Finally, these MEMs devices also have difficultly in detecting small molecules.
In summary, most of the existing label-free detection technologies rely on the detection of mass of molecules, which are difficult to measure small molecules (with low masses). Examples of these mass-sensitive detection technologies include microfabricated mechanical resonators using micro-cantilevers, quartz crystal tuning fork and other mechanical structures. These technologies measure the resonance frequencies of the resonators by detecting harmonic or higher harmonic modes. Since the resonance frequency of a resonator decreases with the mass, these resonator technologies measure molecules by accurately tracking the resonance frequencies. However, the sensitivities of these technologies diminish with the mass of the molecule under detection. Instead of detecting mass, the present embodiments measure charge of molecules. A major advantage of the charge-based detection is that the output signal does not diminish with the size of the molecule, allowing for the detection of both large and small molecules.
The key component of CSOD is an array of optical fiber probes. Each fiber probe is dipped into a well in a standard microplate, and an electric field is applied perpendicular to the fiber. If charge is present on the fiber, the fiber will bend under the electrostatic force. By measuring the amount of bending, one could in principle detect the charge of the fiber, and change of the charge when charged molecules bind to the fiber. However, this design has a serious noise/drift issue due to temperature fluctuations, mechanical stress and noise, and surface tension. One way to overcome this issue is to increase the applied electric field, but associated with the increased field is electrochemical reactions taking place on the electrodes in the well. The reactions change the chemical composition and pH of the solution in the well, and often lead to gas bubble formation (hydrogen and oxygen gasses from water hydrolysis); both are problematic for the detection of molecules.
To combat these problems, embodiments described herein modulate the applied electric field at a frequency and detect the fiber movement at the frequency of the applied electric field. The alternating electric field minimizes the electrochemical reactions, and allows the use of Fourier filer and other noise reduction methods to remove the noise and drift issue.
Unlike the mechanical resonators that rely on the detection of resonance frequency, the present fiber is substantially damped, and the fiber movement is determined by the charge on the fiber and viscous damping of the fiber by the solution.
The frequency of the modulating electric field is preferably to be a few Hz to a few kilo-Hz. Lower frequencies do not provide sufficient removal of the electrochemical reactions and noise/drift. Higher frequencies require fast optical detection, which adds cost to the detection technologies. However, high frequencies may be implanted especially if one shrinks the optical fiber to micron or small dimensions.
Sensitive measurement of the oscillation amplitude can be achieved using a differential optical detection. A straightforward way to implement this detection strategy is to image the optical fiber tip with a CMOS imager or CCD (FIG. 6). The image of the probe tip, as shown in FIG. 2c, appears as a bright spot, which is divided into two regions, A and B, as shown in FIG. 2d. The division is selected such that the intensities in regions A and B are similar initially, and then the differential signal, (IA−IB)/(IA+IB) is monitored continuously with the imager, where IA and IB are the intensities of regions, A and 13, respectively. We have shown that (IA−IB)/(IA−IB) is proportional to the fiber tip displacement. This differential detection method is sensitive because it rejects common-mode noise. In addition to differential optical detection, Fourier filter is used to further remove noise.